CN112105353A

CN112105353A - Methods of treating solid tumors with CCR2 antagonists

Info

Publication number: CN112105353A
Application number: CN201980017482.2A
Authority: CN
Inventors: J·J·坎贝尔; R·辛格; 黄震宇; 吴雪松
Original assignee: University of California; Chemocentryx Inc
Current assignee: University of California; Chemocentryx Inc
Priority date: 2018-01-08
Filing date: 2019-01-07
Publication date: 2020-12-18
Anticipated expiration: 2039-01-07
Also published as: EP3737367A4; US11154556B2; US20190209573A1; CN112105353B; MA50423A; WO2019136368A2; CA3087765A1; AU2019205784A1; US20220241290A1; IL275898A; TW201938160A; EP3737367A2; JP7442141B2; WO2019136368A3; JP2021509678A

Abstract

The present disclosure provides, inter alia, methods of treating solid tumors by administering an effective amount of a chemokine receptor 2(CCR2) antagonist. The invention also provides a method of reducing macrophage numbers in a solid tumor microenvironment, comprising administering a solid tumor vaccine comprising administering a peptide having a peptide structureAn effective amount of a chemokine receptor 2(CCR2) antagonist. In another aspect, the present disclosure further provides a method of increasing the number of CD8+ T cells in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist. In some embodiments, the CCR2antagonist has the structure shown in formula I:

Description

Methods of treating solid tumors with CCR2 antagonists

cross Reference to Related Applications

Priority of U.S. application serial No. 62/614,923 filed 2018, 1, 8,35 u.s.c. § 119(e), the disclosure of which is incorporated herein by reference in its entirety.

Federal-supported research and development claims relating to patent rights

Not applicable.

Reference to a "sequence Listing", a form or a computer program List appendix submitted on an optical disc

Not applicable.

Background

Tumor-associated macrophages (TAMs) are abundant in tumor tissues and can enhance pro-cancerous inflammation [1-3 ]. TAMs promote immunosuppressive Tumor Microenvironments (TMEs) by secreting a number of chemokines essential for the recruitment of immunosuppressive cells. In addition, they produce angiogenic factors (e.g., VEGF), platelet-derived growth factor, and transforming growth factor β to induce neovascularization. Furthermore, PD-L1 (also known as B7H1) on macrophages confers direct inhibitory function to TAMs by inducing antigen-specific tolerance in tumor-bearing hosts [3-5 ].

The abundance of macrophages in TME and their negative association with survival are frequently reported in malignancies (including prostate, breast, colorectal, pancreatic and lymphoma) [4, 6 ]. High macrophage density in tumors is associated with poor patient prognosis and treatment resistance, which also motivates cancer treatment strategies against TAMs [7 ]. Notably, the presence of TAMs in human non-hodgkin lymphoma is not only associated with patient survival, but also with response to treatment [8 ]. Macrophage colony stimulating factor 1receptor (CSF1R) -mediated signaling directs monocyte survival and macrophage differentiation [9 ]. However, clinical trials using the CSF1R blocking strategy were not consistent in showing patient improvement. The main reason for the incomplete inhibition of CSF1R may be due to the dependence of the drug on the entry capacity of malignant cells in TME, thus potentially reducing the therapeutic effect of CSF1R blockers [7, 10-12 ].

Prevention of monocyte recruitment to tumors by targeting the CCL2-CCR2 axis provides another promising strategy [13 ]. Neutralizing CCL2 antibody has been shown to slow tumor progression in preclinical studies [14 ]. However, clinical trials have shown limited clinical response. Pharmacokinetic data show that when monoclonal CCL2 antibody is used to target the CCL2/CCR2 axis in metastatic prostate cancer, the antibody dissociates rapidly and serum CCL2 concentration increases unexpectedly [15-18 ].

In view of the above limitations, CCR2 antagonists are becoming of increasing interest in targeting the CCL2-CCR2 axis [19, 20 ]. In phase 1b studies, oral administration of a small molecule CCR2antagonist (PF-04136309) to block CCR2 showed reduced TAM infiltration and a reduced endogenous anti-tumor immune response to Pancreatic Ductal Adenocarcinoma (PDAC) [21 ]. Overall, clinical trials lacking beneficial results, further studies are needed to quantify the impact on different cancers. Mechanistically, how CCR2 antagonists remodel TME and CCR2 antagonists activate anti-tumor immunity remains to be described in preclinical tumor models of cellular immunomodulation. Furthermore, optimized options and combinations of standard chemotherapy and radiotherapy or immunotherapy for TAM targeting are yet to be developed [22 ].

The present disclosure addresses these needs and provides related advantages as well.

Disclosure of Invention

In one aspect, the present disclosure provides methods of treating solid tumors comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

In some embodiments, the tumor is a lymphoma. In some embodiments, the lymphoma is cutaneous T-cell lymphoma (CTCL).

In another aspect, the present disclosure provides a method of reducing macrophage numbers in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

In another aspect, the present disclosure provides a method of increasing the number of CD8+ T cells in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

In some embodiments, the CCR2 receptor antagonist has the structure shown in formula I

Wherein each variable is as described later.

In some embodiments, the CCR2antagonist has a structure selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structure shown below

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structure shown below

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structure shown below

Or a pharmaceutically acceptable salt thereof.

Drawings

1A-C in the MBL2/DNFB mouse model, oral administration of Compound 1 inhibited tumor growth. (A) The treatment scheme is shown. Compound 1 was fed orally at 20 or 60mg/kg daily for two weeks. Mice were euthanized on day 15 to examine ear tumors. Other mice were euthanized on day 3 or day 7 to determine earlier treatment responses. (B) Examination of ear tumors in MBL2/DNFB mice treated with two different doses of Compound 1 and vehicle for two weeks. A representative ear from each group (8) is shown. (C) On day 15 after euthanasia, the thickness and weight of the ears were measured immediately (p.ltoreq.05, p.ltoreq.001).

Figure 2A-b. oral administration of compound 1 in mice is dose-dependent absorbed and well tolerated. (A) Compound 1 was administered orally daily for ten consecutive days at a lower concentration (20mg/kg daily) or a higher concentration (60mg/kg daily). Plasma concentrations of compound 1 were measured by a chemical analyst 24 hours after the last dose. (B) The body weight of the same group of mice in group (a) was recorded before the first oral administration and after the last oral administration (n-4 per group). Statistical analysis was performed by two-way ANOVA in GraphPad PRISM (GraphPad software, san Diego, Calif.).

Figure 3A-c. compound 1 specifically targets macrophages, but not neutrophils. (A, B) macrophages determined by flow analysis of CD11B +/F4/80+ cell populations in the ear TME (doses indicated in the figure, p <.05,. p <.01, relative to vehicle control) are expressed as percentages or absolute numbers after only two daily doses of Compound 1. (C) Staining of single cell suspensions from the same tissues as (A) with antibodies against CD11b, F4/80, CCR2, Ly6G and Ly 6C. Gated cells on CD11b were further analyzed to differentiate subpopulations. Filled circles represent cells targeted by compound 1. Dashed circles indicate cells that are not blocked by compound 1.

Fig. 4A-c. enhanced inflammation in tumor microenvironment was associated with CCR2 antagonism by compound 1. (A, B) ear tissues from mice treated with Compound 1(60mg/kg) or vehicle were collected on day 7. HE sections of ear tissue from compound 1 and vehicle treated groups and representative images of IHC staining with anti-F4/80 are shown. (C) The ear on day 7 was also analyzed by flow cytometry to quantify the number of two major myeloid subsets in TME with antibodies against CD11b, F4/80, and Ly 6G.

Figure 5A-c. compound 1treatment altered the expression of cytokines and biomarkers produced by TME. Quantitative RT-PCR was performed on ear tissues collected on day 7. Genes involved in cancer inflammation and immune crosstalk (crosstalk) are selectively detected. Comparative expression was performed between compound 1 and vehicle-treated groups. (A) Immunostimulatory cytokines and cytotoxic activation markers are shown; (B) shows pro-inflammatory cytokines; and (C) shows neutrophil chemoattractants (chemoattractrants) and biomarkers. Gene expression values were normalized to the endogenous expression of GAPDH (. p <.05,. p <. 01; n ═ 3 mice per group).

Figure 6 compound 1treatment altered the expression of cytokines and biomarkers produced by TME. Quantitative RT-PCR was performed on ear tissues collected on day 7. Genes involved in immunosuppression and anti-inflammation were selectively detected. Comparative expression was performed between compound 1 and vehicle-treated groups. Gene expression values were normalized to the endogenous expression of GAPDH (n ═ 3 mice per group).

Fig. 7A-e.cd8T cells are forced in CCR2 antagonist-mediated anti-tumor immunity. (A) Two weeks after treatment, tumor tissue from euthanized mice was collected. The groups are as shown. IHC staining was performed with CD8a antibody. The number of CD8 positive T cells was counted by taking three random HPF (high power field) images of each section of four mice per group. (B) Protocol for neutralization of CD8T cells with compound 1 treatment. Neutralizing anti-CD 8 or rat-IgG 2a was administered by intraperitoneal injection one day before MBL2 tumor inoculation, followed by administration of a second dose 7 days later. Tumor formation was detected within two weeks after treatment with compound 1 or vehicle. (C) Mice treated in experiment (B) were euthanized on day 3 for flow analysis of cervical draining lymph nodes to determine the effect of CD8 depletion (three mice per group). (D) Two weeks after compound 1/vehicle treatment (with or without CD8T cell neutralization), mice were euthanized on day 15. The thickness of the ear was measured to show the size of the tumor. (E) Draining lymph nodes were also measured for lymph node metastasis (n-4).

Fig. 8A-d compound 1 and anti-PD 1 have synergistic anti-tumor effects in MBL2 tumors. (A) qRT-PCR was performed to compare the expression of PD1 and PD-L1 in MBL2 tumors formed in ear skin and MBL2 cells cultured in vitro, respectively. (B) anti-PD 1 in combination with compound 1 or vehicle control. (C) Two weeks after treatment, mice spleens were single cell suspension treated and then subjected to intracellular staining for flow analysis (representative flowsheet for each group is shown). (D) Two weeks after treatment, euthanized mice were examined for ear tumors. Representative ear photographs of each group are shown (. p <.05, each group of n ═ 8 mice). The dashed line in the ear thickness bar graph represents the line separating the positive and negative ability to tumor formation.

FIG. 9A-B anti-PD-L1 inhibited the growth of MBL2/DNFB tumors in the ears of mice. anti-PD-L1 (BioXcell, 150ug per mouse, 3 times weekly by IP) was administered starting on the same day as the MBL2 tumor transplantation. Two weeks after treatment, mice were euthanized. The size and weight of the ear tumor was recorded. (A) Photographs of two groups of mice treated with anti-PD-L1 or PBS control are shown. (B) The thickness and weight of the ears were measured (. p <.05,. p <.01, n ═ 4 mice per group).

Detailed Description

I. General purpose

To some extent, the present disclosure is an unexpected and unexpected discovery that CCR2 antagonists can be used to effectively treat solid tumors and related lymphomas.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of T-cell tumors that are predominantly distributed on the skin, including two of the most common types: mycosis Fungoides (MF) and Sezary Syndrome (SS) [23 ]. Signs of skin inflammation are common in CTCLs [24, 25 ]. In diseased skin of MF or SS, the number of CD 163-positive macrophages increases, and the macrophage-expressed CC chemokine ligand 18 promotes a T-helper (Th) 2-dominated microenvironment by inducing chemotaxis of Th2 cells. Such tumor microenvironment is considered a determinant of the progressive clinical behavior of CTCL [26, 27 ]. By targeting TAMs in TME with CCR2 antagonists, an alternative strategy is provided for patients with CTCL at the tumor stage (where good treatment options are limited).

It has previously been reported that a model of higher-order T cell lymphoma was established in mouse skin by injecting MBL 2T lymphoma cells in the skin of the ear followed by the application of 2, 4-dinitro-1-fluorobenzene (DNFB) [28 ]. Tumor formation in this model is strictly dependent on the topical application of DNFB, which triggers an inflammatory skin response, thereby promoting tumor formation. It is demonstrated herein that the small molecule CCR2antagonist compound 1 depletes macrophages in the TME in the ear, resulting in the production of significantly more anti-tumor cytokines (e.g., IFN- γ). Administration of a CCR2antagonist results in expansion of CD8T cells and thus reduced growth of transplanted tumor cells. This mechanism is supported by the following findings: this anti-tumor effect can be counteracted by the simultaneous administration of a neutralizing CD8 monoclonal antibody. Finally, we demonstrate that the therapeutic effect of CCR2 antagonists can be improved by co-administration of an anti-PD 1 antibody. In summary, the present invention demonstrates that preventing the recruitment of TAMs into TMEs may be an effective strategy for treating T cell lymphomas and more generally solid tumors.

Abbreviations and Definitions

CTCL (cutaneous T cell lymphoma); MF (mycosis fungoides); tumor Microenvironment (TME); DNFB (2, 4-dinitro-1-fluorobenzene); PD-1 (programmed death ligand 1); qRT-PCR (quantitative real-time PCR); TAM (tumor associated macrophages); IP (intraperitoneal); mAb (monoclonal antibody); IHC (immunohistochemistry).

Unless otherwise indicated, the term "alkyl", by itself or as part of another substituent, means having the indicated number of carbon atoms (i.e., C)_1-8Refers to a straight or branched chain hydrocarbon group of 1 to8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated hydrocarbon group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated hydrocarbon group having one or more triple bonds. Examples of such unsaturated alkyl groups include ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "cycloalkyl" refers to a ring having the indicated number of ring atoms (e.g., C)_3-6Cycloalkyl) and fully saturated or having no more than one double bond between the ring vertices. "cycloalkyl" may also refer to bicyclic hydrocarbon rings and polycyclic hydrocarbon rings, e.g., bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heterocycloalkyl group can be a monocyclic, bicyclic, or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include: pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. The heterocycloalkyl radical may be attached to the ring carbon or the hetero atomThe other parts of the children are connected. By terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that the cycloalkyl or heterocycloalkyl group is attached to the rest of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl-is a cyclobutyl ring attached to a methylene linking arm and to the rest of the molecule.

The term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, e.g., -CH₂CH₂CH₂CH₂-. Generally, alkyl (or alkylene) groups have 1 to 24 carbon atoms, and those having 10 or less carbon atoms are preferred in the present invention. "lower alkyl" or "lower alkylene" is a short chain alkyl or alkylene group typically having four or fewer carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to the unsaturated forms of "alkylene" having double or triple bonds, respectively.

Unless otherwise specified, the term "heteroalkyl," by itself or in combination with another term, refers to a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and one to three heteroatoms selected from O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S can be located anywhere within the heteroalkyl group. The heteroatom Si may be located anywhere in the heteroalkyl group, including the position where the alkyl group is attached to the remainder of the molecule. Examples include: -CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂,-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-Si(CH₃)₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, e.g. -CH2-NH-OCH₃and-CH₂-O-Si(CH₃)₃. Similarly, unless otherwise indicated, the term "heteroalkeneThe groups "and" heteroalkynyl "by themselves or in combination with another term, mean alkenyl or alkynyl radicals containing the recited number of carbons and having one to three heteroatoms selected from O, N, Si and S, respectively, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S can be located anywhere within the heteroalkyl group.

The term "heteroalkylene" by itself or as part of another substituent refers to a divalent saturated or unsaturated or polyunsaturated radical derived from a heteroalkyl radical, e.g., -CH₂-CH₂-S-CH₂CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-、-O-CH₂-CH＝CH-、-CH₂-CH＝C(H)CH₂-O-CH₂-and-S-CH₂-C ≡ C-. For heteroalkylene groups, heteroatoms can also be located at one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamine, and the like).

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an ammonia atom or a sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties can be the same or different and can be attached to the 3-7 membered ring through the nitrogen atom to which each is attached. Thus, with NR^aR^bGroups represented include piperidinyl, pyrrolidinyl, morpholinyl, azadinyl, and the like.

Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C_1-4Haloalkyl "is meant to include trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

Unless otherwise indicated, the term "aryl" refers to a polyunsaturated, usually aromatic, hydrocarbon group that can be a single ring or multiple rings (up to three rings) that are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. Heteroaryl groups may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include: phenyl, naphthyl, and biphenyl; and non-limiting examples of heteroaryl groups include: pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzpyrazolyl, benzoxazolyl (benzoxazolyl), benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyridyl, thienopyrimidinyl, imidazopyridyl, benzothiazolyl (benzoxaxol), benzofuranyl, benzothiophenyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like. The substituents for each of the aryl and heteroaryl ring systems described above are selected from the group of acceptable substituents described hereinafter.

For the sake of brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthio, arylalkyl) includes aryl and heteroaryl rings as defined above. Thus, the term arylalkyl is intended to include groups in which the aryl group is attached to an alkyl group that is attached to the remainder of the molecule (e.g., benzyl, phenethyl, pyridylmethyl, and the like).

In some embodiments, the above terms (e.g., alkyl, aryl, and heteroaryl) are intended to include both substituted and unsubstituted forms of the indicated groups. Preferred substituents for each group are provided below. For the sake of brevity, the terms aryl and heteroaryl will refer to the substituted or unsubstituted forms provided below, while the term "alkyl" and related aliphatic groups refer to the unsubstituted forms, unless otherwise indicated.

Alkyl (including what is commonly referred to as alkylene)Those of alkenyl, alkynyl and cycloalkyl) substituents may be a number of from zero to (2m' +1) of various groups selected from the group consisting of: halogen, OR ', NR' R ', SR', -SiR 'R', -OC (O) R ', C (O) R', -CO₂R'、-CONR'R”、-OC(O)NR'R”、-NR”C(O)R'、-NR'-C(O)NR”R”'、-NR”C(O)₂R'、-NH-C(NH₂)＝NH、-NR'C(NH₂)＝NH、-NH-C(NH₂)＝NR'、-S(O)R'、-S(O)₂R'、-S(O)₂NR'R”、-NR'S(O)₂R ", -CN, and-NO₂Wherein m' is the total number of carbon atoms in the group. R ', R ' and R ' each independently mean hydrogen, unsubstituted C_1-8Alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C_1-8Alkyl radical, C_1-8Alkoxy or C_1-8Thioalkoxy or unsubstituted aryl-C_1-4An alkyl group. When R' and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a3, 4,5, 6 or 7 membered ring. For example, -NR' R "is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl" used alone or as part of another group means that two substituents on the carbon closest to the point of attachment of the group in an alkyl group are substituted with a substituent ═ O (e.g., -c (O) CH₃、-C(O)CH₂CH₂OR', etc.).

Similarly, the substituents for aryl and heteroaryl groups are variable and are typically selected from: -halogen, -OR ', -OC (O) R ', -NR ' R ", -SR ', -R ', -CN, -NO₂、-CO₂R'、CONR'R”、C(O)R'、-OC(O)NR'R”、-NR”C(O)R'、-NR”C(O)₂R'、-NR'-C(O)NR”R”'、NHC(NH₂)＝NH、-NR'C(NH₂)＝NH、-NH-C(NH₂)＝NR'、-S(O)R'、-S(O)₂R'、S(O)₂NR'R”、NR'S(O)₂R”、N₃Perfluoro (C)₁-C₄) Alkoxy and perfluoro (C)₁-C₄) Alkyl groups in a number ranging from zero to the total number of open valences on the aromatic ring system; and R ', R ", and R'" are independently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl, unsubstituted aryl and heteroAryl, (unsubstituted aryl) -C_1-4Alkyl, and unsubstituted aryloxy-C_1-4An alkyl group. Other suitable substituents include the various aryl substituents described above attached to the ring atoms through an alkylene group of 1 to 4 carbon atoms.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted by a group of formula-T-C (O) - (CH)₂)_q-U-substituent, wherein T and U are independently-NH-, -O-, -CH₂-or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by a group of formula-A- (CH)₂)_rA substituent of-B-, wherein A and B are independently-CH₂-、-O-、-NH-、-S-、-S(O)-、-S(O)₂-、-S(O)₂NR' or a single bond, and r is an integer of 1 to 3. One of the single bonds of the new ring so formed may be optionally substituted by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted by a group of formula- (CH)₂)_s-X-(CH₂)_t-substituent (S) wherein S and t are independently an integer from 0 to 3, and X is-O-, -NR' -, -S (O)₂-, or-S (O)₂NR' -. At NR' -and-S (O)₂The substituents R 'in NR' are selected from: hydrogen or unsubstituted C_1-6An alkyl group.

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

For the compounds provided herein, a bond extending from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) is understood to mean a bond that provides attachment at any available vertex position of the aromatic ring. In some embodiments, the description will also include a connection on a ring fused to the aromatic ring. For example, a bond extending from the center of the benzene ring of an indole represents a bond to any available vertex of a six-or five-membered ring moiety of an indole.

The term "pharmaceutically acceptable salt" is intended to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents present on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired acid, which may be solvent-free or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as salts derived from relatively nontoxic organic acids such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are Salts of amino acids such as arginine and the like, and Salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge, s.m., et al, "pharmaceutically acceptable Salts", Journal of Pharmaceutical Science (1977, 66, 1-19). Certain specific compounds of the invention contain both basic and acidic functionalities that allow the compounds to be converted into base or acid addition salts.

The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt is equivalent to the parent form of the compound for purposes of the present invention.

In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that are susceptible to chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to a compound of the invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical agent.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polycrystalline or amorphous form. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (e.g., individual enantiomers) are intended to be included within the scope of the present invention. When compounds having a defined stereochemistry (denoted as R or S, or indicated by a dashed or wedged bond) are provided herein, it will be understood by those skilled in the art that these compounds will be substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99%, and up to 100% free of other isomers).

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. Unnatural proportions of isotopes can be defined from amounts found in nature to 100% of the amount made up of the atoms in question.For example, the compounds may incorporate radioactive isotopes (e.g., tritium(s) (iii))³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) Or a non-radioactive isotope (e.g. deuterium: (ll))²H) Or carbon-13 (¹³C) ). Such isotopic variations may provide additional utility to those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find other uses, including but not limited to, as diagnostic and/or imaging agents, or as cytotoxic/radiotoxic therapeutic agents. In addition, isotopic variations of the compounds of the present invention can have altered pharmacokinetic and pharmacodynamic profiles that can contribute to improved safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

As used herein, the term "solid tumor" refers to a malignant tumor. Solid tumors are typically localized tissue masses; however, solid tumors are able to invade surrounding tissues and migrate to the new body side. Solid tumors may be benign (non-cancer) or malignant (cancer). Different types of solid tumors are named for the cell types that form them. Examples of solid tumors are sarcomas, carcinomas (carcinomas) and lymphomas. The term "solid tumor" does not include leukemia (leukemia). A "sarcoma" is a cancer caused by connective or supportive tissue (e.g., bone or muscle). "cancer (Sarcomas)" is a cancer caused by glandular cells and epithelial cells arranged in human tissues. A "lymphoma" is a cancer of lymphoid organs (e.g., lymph nodes, spleen, and thymus). Since these cells are present in most tissues of the human body, lymphoma may occur in various organs. Exemplary solid tumors include, but are not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma (endothieliosarcoma), lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, testicular carcinoma, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioblastoma multiforme (gliobastoma), Astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, cutaneous T-cell lymphoma (CTCL), melanoma, neuroblastoma, and retinoblastoma.

Detailed description of the preferred embodiments

A. Method of producing a composite material

In some embodiments, the solid tumor is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilm's tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, or a combination thereof, Hemangioblastoma, acoustic neuroma, oligodendroglioma, cutaneous T-cell lymphoma (CTCL), melanoma, neuroblastoma, and retinoblastoma.

In some embodiments, the solid tumor is a brain cancer, breast cancer, triple negative breast cancer, bladder cancer, bone cancer, colorectal cancer, lung cancer, kidney cancer, liver cancer, stomach cancer, prostate cancer, sarcoma, melanoma, carcinoma, or lymphoma.

In some embodiments, the solid tumor is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, or lymphoma.

In some embodiments, the solid tumor is a lymphoma. In some embodiments, the lymphoma is cutaneous T-cell lymphoma (CTCL). As mentioned above, Cutaneous T Cell Lymphoma (CTCL) is a heterogeneous T cell tumor located predominantly in the skin.

The CTCL is typically divided into four separate phases (including sub-phases). Early CTCL (stages IA and IB) involves the skin being covered by red patches (patches) or plaques (plaques). The difference between phase IA and IB is the amount of skin affected by the red patches or plaques. At stage IIA, except for skin patches/plaques, the affected individual has enlarged lymph nodes, but the cancer has not spread to the lesion. Stage IIB is the stage where one or more tumors are found on the skin (i.e., "tumor stage CTCL"), and the lymph nodes may be swollen, but the cancer has not spread to the lymph nodes. In stage III CTCL, almost all skin turns red, including patches, plaques, and/or tumors, lymph nodes may be swollen, but the cancer has not spread to the lymph nodes. In stage IV, the cancer spreads to lymph nodes or other organs.

The present disclosure contemplates treating any stage in stages I-IV with the methods described herein. In some embodiments, the subject is early CTCL (i.e., stage IA, IB, or IIA). In some embodiments, a subject with CTCL is in stage IIB or later (i.e., "tumor-stage CTCL"). Thus, in some embodiments, the subject is diagnosed with stage IIB or more advanced CTCL. In some embodiments, the subject is diagnosed with stage IIB CTCL.

In some embodiments, the CTCL is a specific subtype of CTCL. In some embodiments, the CTCL is Mycosis Fungoides (MF). In some embodiments, the CTCL is Sezary Syndrome (SS).

In a second aspect, the invention provides a method of reducing macrophage numbers in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

In a third aspect, the invention provides a method of increasing the number of CD8+ T cells in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

CCR2 antagonists

In some embodiments, the CCR2antagonist is a small molecule antagonist of CCR2 having the formula (I):

or a pharmaceutically acceptable salt, hydrate, stereoisomer, or rotamer thereof; wherein

A is C (R)⁵)(R⁶) Or N (R)⁵)

Subscripts m and n are each independently an integer of from 0 to 2, and m + n.ltoreq.3;

R¹selected from the group consisting of: aryl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl, wherein the heteroaryl moiety has 1 to 3 moieties selected from: n, O and a heteroatom of S as a ring member; and wherein said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 5R^xSubstituent group substitution;

R²selected from the group consisting of: H. c_1-8Alkyl radical, C_3-8Cycloalkyl radical, C_3-8cycloalkyl-C_1-4Alkyl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl, wherein the heteroaryl moiety has 1-3 heteroatoms selected from N, O and S as ring members; wherein said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 4R^xSubstituent group substitution;

or optionally, R¹And R²Combined with the nitrogen atom to which it is attached to form a 6-to 11-membered monocyclic or fused bicyclic heterocyclic or heteroaryl ring, wherein-NR¹R²Optionally further substituted with 1 to 4R^xSubstituent group substitution;

R³selected from the group consisting of: H. c_1-8Alkyl radical, C_3-8Cycloalkyl and C_3-8cycloalkyl-C_1-4Alkyl, each of which is optionally substituted with 1-3R^ySubstituent group substitution;

R⁴selected from the group consisting of: H. optionally substituted by 1 to 2R^ySubstituted C_1-8Alkyl and-CO₂H：

R⁵Selected from the group consisting of: c_1-8Alkyl radical, C_1-8Alkoxy radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkoxy, C_3-8cycloalkyl-C_1-4Alkyl radical, C_1-8Alkylamino radical, di-C_1-8Alkylamino, aryl, aryloxy, arylamino, aryl-C_1-4Alkyl, heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C_1-4Alkyl, each of which is optionally substituted with 1 to 5R^zSubstituent group substitution;

R⁶selected from the group consisting of: H. f, OH, C_1-8Alkyl and C_1-8Alkoxy radical, wherein, C_1-8Alkyl and C_1-8Alkoxy groups optionally substituted with 1 to 3R^zSubstituent group substitution;

or optionally, R⁵And R⁶Combined to form a spirocyclic 5-or 6-membered cycloalkyl ring, which may optionally be unsaturated, and having optionally substituted 1 to 4R^zA fused aryl group substituted with a substituent;

each R^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aC(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-NR^aR^b、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅、-S(O)₂NR^aR^bAnd 5-or 6-membered aryl or heteroaryl, wherein each X¹Is C_1-4An alkylene group; each R^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^aAnd R^bMay be combined with a nitrogen atom to form a 5-or 6-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by oxo; each R^cIndependently selected from: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; optionally, and when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five or six membered carbocyclic ring, and wherein the aryl or heteroaryl group is optionally substituted with 1 to 3 members selected from the group consisting of: halogen, hydroxy, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Haloalkyl and C_1-4A haloalkoxy group;

each R^yIndependently selected from the group consisting of: halogen, -CN, -R^f、-CO₂R^d、-CONR^dR^e、-C(O)R^d、-OC(O)NR^dR^e、-NR^eC(O)R^d、-NR^eC(O)₂R^f、-NR^dC(O)NR^dR^e、-NR^dC(O)NR^dR^e、-NR^dR^e、-OR^dand-S (O)₂NR^dR^e(ii) a Wherein each R is^dAnd R^eIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^dAnd R^eMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members; each R^fIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group;

each R^zIndependently selected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-OC(O)NR^gR^h、-NR^hC(O)R^g、-NR^hC(O)₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-S(O)₂NR^gR^h、-X¹-R^j、-X¹-NR^gR^h、-X¹-CONR^gR^h、-X¹-NR^hC(O)R^g、-NHR^j、-NHCH₂R^jAnd tetrazole; wherein each R is^gAnd R^hIndependently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^gAnd R^hMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by one or two oxo groups; each RⁱIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; each R^jSelected from the group consisting of: c_3-6Cycloalkyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl and tetrahydropyranyl.

It should be understood that when R is¹And R²When combined with the nitrogen atom to which each is attached to form a 6-to 11-membered monocyclic or fused bicyclic heterocycle, the 6-to 11-membered monocyclic or fused bicyclic heterocycle includes a monocyclic heterocycle fused to an aryl or heteroaryl ring.

In formula I, in one embodiment, the substituent R³Selected from the group consisting of: H. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl and cyclobutylmethyl.

In the description herein, one skilled in the art will understand that the wavy line intersecting a bond is intended to define the point of attachment of a substituent or group to the rest of the molecule.

As noted above, subscripts m and n are integers selected from 0, 1 and 2, respectively, and m + n ≦ 3. When the subscript is 0, one skilled in the art will understand that a cyclic structure having ring vertex a is contemplated, but adjacent ring vertices on the other side of the parenthesis are joined by a key. The present invention therefore includes those structures in which the ring with a as the vertex comprises:

in a selected set of embodiments, m and n are both 0. In another selected group of embodiments, m and n are both 1. In yet another selected group of embodiments, m is 1 and n is 0. In yet another set of embodiments, m is 1 and n is 2.

In other selected embodiments, the ring having vertex a is represented by an equation selected from the group consisting of:

in a subgroup of embodiments, the compound of formula (I) is represented by the formula:

within formula (Ia), a number of selected embodiments are provided, as shown in formulas Ia1, Ia2, Ia3, Ia4, and Ia 5.

In the respective formulae Ia, Ia1, Ia2, Ia3, Ia4 and Ia5, the substituent (R)¹To R⁶、R^xAnd R^z) And subscripts m and n have the meanings provided above with respect to formula I. The subscripts p and q have the following meanings: for Ia1, Ia4, and Ia5, subscript q is an integer of 0 to 5; for Ia2 and Ia4, subscript p is an integer from 0 to 4; for Ia3 and Ia5, subscript p is an integer of 0 to 5.

In other selected embodiments, the compounds provided herein are represented by a formula selected from:

wherein each compound is substantially free of other stereoisomers, and wherein the substituent (R)¹To R⁶、R^xAnd R^z) And subscripts m and n have the meanings provided above with respect to formula I. The subscripts p and q have the following meanings: for Ia1', Ia4', and Ia5', subscript q is an integer of from 0 to 5; for Ia2 'and Ia4', subscript p is an integer of from 0 to 4; for Ia3 'and Ia5', subscript p is an integer of from 0 to 5.

In another group of embodiments of formula I, A is C (R)⁵)(R⁶) Wherein R is⁵And R⁶The binding forms a ring. Selected embodiments are as follows:

in each of the formulae Ib, Ib1 and Ib2, the substituent (R)¹To R⁶、R^xAnd R^z) And subscripts m and n have the meanings provided above with respect to formula I. The subscripts p and q have the following meanings: for Ib, Ib1 and Ib2, subscript q is an integer from 0 to 5; for Ib1, subscript p is an integer from 0 to 4; for Ib2, subscript p is an integer from 0 to 5.

In another group of embodiments of formula I, A is NR⁵(see formula Ic). Selected embodiments are as follows:

in each of formulae Ic, Ic1, Ic2, Ic3, Ic4 and Ic5, the substituent (R)¹To R⁶、R^xAnd R^z) And subscripts m and n have the meanings provided above with respect to formula I. The subscripts p and q have the following meanings: for Ic1, Ic4, and Ic5, subscriptsq is an integer of 0 to 5; for Ic2 and Ic4, subscript p is an integer from 0 to 4; for Ic3 and Ic5, subscript p is an integer from 0 to 5.

wherein each compound is substantially free of other stereoisomers, and wherein the substituents (R1 to R6, Rx and Rz) and the subscripts m and n have the meanings provided above for formula I. The subscripts p and q have the following meanings: for Ic 1', Ic 4', and Ic 5', the subscript q is an integer from 0 to 5; for Ic2 'and Ic 4', subscript p is an integer from 0 to 4; for Ic3 'and Ic 5', subscript p is an integer from 0 to 5.

In other selected embodiments, compounds of formula I, Ia1, Ia1', Ib, Ic1, and Ic 1', each as described above, are provided, wherein, -N (R) is¹)(R²) Selected from:

in other selected embodiments, there are provided compounds of formula I, Ia1, Ia1', Ib, Ic1, and Ic 1', each as described above, wherein, -N (R)¹)(R²) Selected from:

in some embodiments, compounds of formula I, Ia2, Ia3, Ia2', and Ia3' are provided wherein a is C (R)⁵)(R⁶) Or of the formula C (R)⁵)(R⁶) In which R is⁵Selected from: aryl, aryloxy, arylamino, aryl-C_1-4Alkyl, heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C_1-4Alkyl, wherein the aryl or heteroaryl moiety or moiety is selected from:

group 1

In certain selected embodiments, compounds of formula I, Ia2, Ia3, Ia2', and Ia3' are provided wherein a is C (R)⁵)(R⁶) Or of the formula C (R)⁵)(R⁶) In which R is⁵Selected from: aryl, aryloxy, arylamine and aryl-C_1-4Alkyl, wherein the aryl group or moiety is selected from:

subgroup 1a

In other selected embodiments, compounds of formula I, Ia2, Ia3, Ia2', and Ia3' are provided wherein a is C (R)⁵)(R⁶) Or of the formula C (R)⁵)(R⁶) In which R is⁵Selected from: heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C_1-4Alkyl, wherein the heteroaryl group or moiety is selected from:

subgroup 1b

In some embodiments, compounds of formula I, Ic2, Ic3, Ic 2', and Ia3' are provided, wherein a is N (R)⁵) Or of the formula N (R)⁵) In which R is⁵Selected from: aryl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl, wherein the aryl or heteroaryl group or moiety is selected from group 1 above. In certain selected embodiments, compounds of formula I, Ic2, Ic3, Ic2 'and Ic 3' are provided wherein a is N (R)⁵) Or of the formula N (R)⁵) In which R is⁵Selected from: aryl and aryl-C_1-4Alkyl, wherein the aryl group or moiety is selected from the above sub-group 1 a. In other selected embodiments, compounds of formula I, Ic2, Ic3, Ic2 'and Ic 3' are provided wherein a is N (R)⁵) Or of the formula N (R)⁵) Wherein, R is⁵Selected from: heteroaryl and heteroaryl-C_1-4Alkyl, wherein the heteroaryl group or moiety is selected from the above sub-group 1 b.

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structural formula shown in compound 1

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structural formula shown in compound 2

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist has the structural formula shown in compound 3

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2antagonist is selected from the group consisting of compounds or pharmaceutical compositions disclosed in US2016/0340356, US2016/0340356 derived from application No. 15/158,713 filed by chemicentryx on 5/19 of 2016, the contents of which are incorporated herein for all purposes.

In some embodiments, the CCR2 chemokine receptor antagonist is selected from the group consisting of: AZ889, AZD2423, INCB-8761, MK-0812, BMS-813160, INCB-003284, PF-04634817, BMS-741672, ceniviroc (Cenicrivroc), CCX-140.

C. Method of administration

The term "therapeutically effective dose" refers to the amount of a compound of interest that will elicit the biological or medical response of a cell, tissue, system or animal (e.g., a human) that is being sought by a researcher, veterinarian, medical doctor or other treatment provider.

In general, the treatment methods provided herein comprise administering to a patient an effective amount of one or more compounds provided herein. In a preferred embodiment, the compounds of the invention are preferably administered orally or topically to a patient (e.g., human). The treatment regimen may vary depending upon the compound used and the particular condition being treated; for the treatment of most conditions, a daily dosing frequency of 4 or less is preferred. In general, a 2-time daily dosage regimen is more preferred, with once-a-day administration being particularly preferred. It will be understood, however, that the specific dose level and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination (i.e., other drugs administered to the patient), and the severity of the particular disease undergoing therapy, and the judgment of the prescribing physician. In general, it is preferred to use a minimum dose sufficient to provide effective treatment. The effectiveness of a treatment in a patient can generally be monitored using medical or veterinary criteria appropriate to the condition being treated or prevented.

Depending on the disease to be treated and the condition of the patient, the compounds and compositions of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical routes of administration, and may be formulated, alone or together, in suitable dosage unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each administration. The invention also contemplates administration of the compounds and compositions of the invention as long acting formulations.

Dosage levels of about 0.1mg to about 140mg per kilogram of body weight per day are useful for treating or preventing conditions (about 0.5mg to about 7g per day per person) in which pathogenic CCR2 activity is implicated. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Unit dosage forms typically contain between about 1mg to about 500mg of the active ingredient. For compounds administered orally, transdermally, intravenously, or subcutaneously, preferably, a sufficient amount of the compound is administered to achieve a serum concentration of 5ng (nanogram)/mL to 10g (microgram)/mL of blood, more preferably, a sufficient amount of the compound is administered to achieve a serum concentration of 20ng to 1 microgram/mL of serum, and most preferably, a sufficient amount of the compound is administered to achieve a serum concentration of 50ng/mL to 200 ng/mL. Sufficient compound should be administered for direct injection into the synovium (for the treatment of arthritis) to achieve a local concentration of about 1 micromolar.

The frequency of dosage may also vary depending on the compound used and the particular disease being treated. However, for the treatment of most conditions, a dosage regimen of 4 times daily, 3 times daily or less is preferred, and a once daily or 2 times daily dosage regimen is particularly preferred. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e., other drugs administered to the patient), the severity of the particular disease undergoing treatment, and other factors including the judgment of the prescribing physician.

In some embodiments, the treatment or prevention of conditions requiring the modulation of CCR2 receptors is indicated, typically at a dosage level of about 0.001 to 100mg per day per kg body weight of the patient, which may be administered in single or multiple doses. Preferably, the dosage level is from about 0.01 to about 25mg/kg per day; more preferably from about 0.05 to about 10mg/kg per day. Suitable dosage levels may be about 0.01 to 25mg/kg per day, about 0.05 to 10mg/kg per day, or about 0.1 to 5mg/kg per day. Within this range the dosage may be 0.005 to 0.05, 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing from 1.0 to 1000 mg of the active ingredient, in particular, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 mg of the active ingredient for symptomatic adjustment of the dose to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, genetic characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease state, and the host undergoing therapy.

D. Combination therapy

In treating, preventing, ameliorating, controlling or reducing solid tumor growth and metastasis, the compounds of the present invention may be used in conjunction with: (1) cancer vaccination strategies, (2) immune checkpoint modulators, e.g. antagonistic antibodies against immune checkpoint inhibitors (anti-PD 1, anti-PD-L1, anti-CTLA 4, anti-Tim 3, anti-VISTA, anti-KIR) or agonistic antibodies against immune promoters (anti-Lag 3, anti-OX 40, anti-ICOS, anti-4-1 BB, (3) blocking or abrogating antibodies against cell surface proteins normally upregulated in transformed cells (CEACAM1, Syndecan) -2, GRP78), (4) anti-angiogenic therapies (anti-VEGF, anti-VEGFR, VEGFR small molecule inhibitors), (5) blocking antibodies (blocking antibodies) or inhibitors against VEGF, FDF2, PDGF and their respective receptors), (6) standard chemotherapeutic therapies (gemcitabine ), Paclitaxel, follorinox), (7) radiotherapy, (8) other chemokine antagonists (CCR1, CCR4, CCR6, CXCR4, CXCR2, CXCR7 small molecule inhibitors, blocking antibodies or depleting antibodies), (9) depleting antibodies against chemokines that activate the above chemokine receptors, (10) inhibitors that target somatic mutations common in cancer, such as those specific for the following genes (BRAF, KRAS, NRAS, EGFR, CTNNB1, NOTCH1, PIK3CA, PTEN, APC, FLT3, IDH1, IDH2, KIT, TP53, JAK 2). Combination therapies are also contemplated in methods of increasing the number of CD8+ T cells in a solid tumor microenvironment and methods of decreasing the number of macrophages in a solid tumor microenvironment.

In some embodiments, the compounds of the present invention may be used in combination with an anti-inflammatory or analgesic agent (e.g., an opiate agonist), a lipoxygenase inhibitor (e.g., an inhibitor of 5-lipoxygenase), a cyclooxygenase inhibitor (e.g., a cyclooxygenase-2 inhibitor), an interleukin inhibitor (e.g., an interleukin-1 inhibitor), an NMDA antagonist, a nitric oxide inhibitor or a nitric oxide synthesis inhibitor, a non-steroidal anti-inflammatory agent, or a cytokine-inhibiting anti-inflammatory agent, for example, in combination with a compound such as acetaminophen, aspirin, codeine, a biological TNF chelating agent, fentanyl, ibuprofen, indomethacin (indomethacin), ketorolac, morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic, fentanyl (sufentanyl), sulindac (sulindac), tenidap (tenidap), and the like.

In some embodiments, the immune checkpoint inhibitor is a PD-1 and/or PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor may be Durvalizumab or Atlizumab or Avelmumab or BMS-936559(MDX-1105) or ALN-PDL or TSR-042 or KD-033 or CA-170 or CA-327 or STI-1014 or MEDI-0680 or KY-1003. Duvacizumab (MEDI4736) is a human monoclonal antibody directed against PD-L1. Attrituximab (MPDL3280A) is a fully humanized, engineered IgG1 monoclonal antibody directed against PD-L1. Avizumab (MSB0010718C) is a fully humanized, engineered IgG1 monoclonal antibody directed against PD-L1. BMS-936559(MDX-1105) is a fully humanized IgG4 monoclonal antibody directed against PD-L1. ALN-PDL is an inhibitory RNA (RNAi) that targets PD-L1. TSR-042 refers to an engineered chimeric antibody against the PD-1/PD-L1 pathway. KD-033 refers to a bifunctional anti-PD-L1/IL-15 fusion protein in which an anti-PD-L1 antibody is linked at its tail to the cytokine IL-15 via the sushi domain of the IL-15 receptor. CA-170 refers to a small molecule antagonist of PD-L1 and VISTA. STI-1014 refers to an anti-PD-L1 antibody. KY-1003 is a monoclonal antibody against PD-L1. CA-327 refers to a small molecule antagonist of PD-L1 and TIM 3.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: duvacizumab, Atlantibizumab, pembrolizumab (pembrolizumab), nivolumab (nivolumab), AP-106, AP-105, MSB-2311, CBT-501, Avermemab, AK-105, IO-102, IO-103, PDR-001, CX-072, SHR-1316, JTX-4014, GNS-1480, recombinant humanized anti-PD 1mAb (Shanghai June bioscience), REGN-2810, perlarcept (pelareorecep), SHR-1210, PD 1/1 inhibitor vaccine (THERAVECTYS), BGB-A St, recombinant humanized anti-PD-1 mAb (Baiotai Biol-Thea Solutions), PDL-1 targeting precursor (CytomX), X20717, FS-118, PSI-001, PD-01, TILTAIM-07, TIPLE-1 modified PDL (Mongolian L-PDL plus therapy Co., Ltd.), PRS-332, FPT-155, Jenno (jienuo) mAb (Jia and biopharmaceutical-Genor Biopharma), TSR-042, REGN-1979, REGN-2810, Renostat (reminostat), FAZ-053, PD-1/CTLA-4 bispecific antibody (MacroGenics), MGA-012, MGD-013, M-7824, PD-1 based bispecific antibody (Beijing Hanmei drug), AK-112, AK-106, AK-104, AK-103, BI-754091, ENUM-244C8, MCLA-145, MCLA-134, anti-PD 1 oncolytic monoclonal antibody (transgenic SA company-Transgene SA), AGEN-2034, IBI-308, WBP-3155, JNJJ-63723283, MEDI-0680, SSI-361, PD-502, anti-TrandBt-1 bispecific antibody, Dual-targeting anti-PD-1/LAG-3 monoclonal antibody (TESARO), dual-targeting anti-PD-1/TIM-3 monoclonal antibody (TESARO), PF-06801591, LY-3300054, BCD-100, STI-1110, pembrolizumab biosimid, nivolumab biosimid, PD-L1-TGF-beta therapy, KY-1003, STI-1014, GLS-010, AM-0001, GX-P2, KD-033, PD-L1/BCMA bispecific antibody (immuno-pharmaceutical), PD-1/Ox40 targeting bispecific antibody (immuno-pharmaceutical), BMS-936559, anti-PD-1/VEGF-A DARPins (Molecular Partners), mDX-400, ALN-PDL, PD-1 inhibitory peptide (aurogen), Auricum-9359, siRNA-loaded dendritic cell vaccines (Alilam Pharmaceuticals), GB-226, PD-L1 Targeted CAR-TNK based immunotherapy (TNK therapeutics/NantKwest), INSIX RA, INDUS-903, AMP-224, anti-CTLA-4/anti-PD-1 bispecific humanized antibodies (Akeso biopharmaceuticals), B7-H1 vaccine (national emphasis laboratory of Oncology/fourth university of military medicine), and GX-D1.

In some embodiments, the PD-1 inhibitor may be pembrolizumab or nivolumab or IBI-308 or mDX-400 or BGB-108 or MEDI-0680 or SHR-1210 or PF-06801591 or PDR-001 or GB-226 or STI-1110. Nivolumab (also called OPDIVO)^TMMDX-1106, BMS-936558 and ONO-4538) are human IgG4 monoclonal antibodies directed against PD-1. Pembrolizumab (also known as

Lambruzumab (lambrolizumab) and MK-34) are humanized IgG4 kappa isotype monoclonal antibodies to PD-1. IBI-308 refers to a monoclonal antibody directed against PD-1. mDX-400 refers to a mouse antibody directed against PD-1. BGB-108 is a humanized monoclonal antibody directed against PD-1. MEDI-0680(AMP-514) is a humanized IgG4 monoclonal antibody directed against PD-1. SHR-1210 refers to a monoclonal antibody against PD-1. PF-06801591 is a monoclonal antibody directed against PD-1. PDR-001 refers to a monoclonal antibody directed against PD-1. GB-226 refers to monoclonal antibodies directed against PD-1. STI-1110 refers to a monoclonal antibody directed against PD-1.

In some embodiments, the PD-1 inhibitor is RPM 1-14.

In some embodiments, the PD-1 inhibitor is an antibody selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab (pidilizumab).

The anti-PD-1 antibodies and antibody fragments described herein include proteins having amino acid sequences different from the antibody but that retain the ability to bind PD-1.

In some embodiments, anti-PD-1 antibodies include bispecific antibodies and antibody-like therapeutic proteins, including those that bind PD-1

Fab derivatives, and the like.

The anti-PD-L1 antibodies and antibody fragments described herein include proteins having amino acid sequences different from the antibody but that retain the ability to bind to PD-L1. Such variant antibodies and fragments thereof may comprise one or more amino acid additions, deletions or substitutions when compared to the parent sequence, but which exhibit substantially equivalent or substantially bioequivalent biological activity to that of the described antibody.

In some embodiments, anti-PD-L1 antibodies include bispecific antibodies and antibody-like therapeutic proteins, including those that bind to PD-L1

Fab derivatives, and the like.

Non-limiting examples of other PD-1/PD-L1 pathway inhibitors are described, for example, in Chen and Han, journal of clinical medicine (journal of Clin Invest), 2015, 125 (9): 3384-3391, U.S. Pat. Nos. 8,168,757; 8,354,509, respectively; 8,552,154, respectively; 8,741,295, respectively; and 9,212,224; U.S. patent application publication numbers 2014/0341917; 2015/0203580 and 2015/0320859; international patent application publication No. WO 2015/026634.

In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. Many CTLA-4 inhibitors are known in the art. In some embodiments, the CTLA-4 inhibitor is an antibody. In some embodiments, the CTLA-4 inhibitor antibody is selected from the group consisting of: ipilimumab (Ipilimumab), Tremelimumab (Tremelimumab), AGEN1884, and AGEN 2041. In some embodiments, the CTLA-4 inhibitor antibody is ipilimumab. In some embodiments, the CTLA-4 inhibitor antibody is tremelimumab. In some embodiments, the CTLA-4 inhibitor antibody is age 1884. In some embodiments, the CTLA-4 inhibitor antibody is age 2041.

If, for example, a biological product (e.g., an antibody or fragment thereof) is highly similar to a biological product that has been approved by the FDA (referred to as a reference), such biological product is considered a biological analog. The biological analogs have no clinical difference from the reference products in terms of safety and effectiveness. The biological analogs may also have the same mechanism of action, route of administration, dosage form and strength as their reference.

Two biologicals (e.g., antibodies or fragments thereof) are considered bioequivalents if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not differ significantly in their rate and extent of absorption when administered in the same molar dose (single or multiple doses) in similar experiments. Certain antibodies will be considered equivalents or drug substitutes if they are equivalent in extent of absorption, but not in rate of absorption and may also be considered bioequivalent because such differences in rate of absorption are intentional and reflected in the markers, are not necessary to achieve effective in vivo drug concentrations as in long-term use and are medically deemed to be immaterial for the particular drug under study.

In some embodiments, two biologies (e.g., two antibodies or fragments thereof) are bioequivalent if they do not differ clinically in safety, purity, or potency.

In other embodiments, two biologicals (e.g., two antibodies or fragments thereof) are bioequivalent if a patient can switch one or more times between a reference and a biologicals without increasing the expected risk of adverse effects, including a clinically significant change in immunogenicity or reduced effectiveness as compared to continued therapy without such switching.

In other embodiments, two biologies (e.g., two antibodies or fragments thereof) are bioequivalent to the extent that such mechanisms are known, if both biologies (e.g., both antibodies or fragments thereof) act through a common mechanism of action under conditions of use.

Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence testing includes, for example, (a) in vivo testing in humans or other mammals, wherein the concentration of an antibody or metabolite thereof (as a function of time) is determined in blood, plasma, serum or other biological fluids; (b) in vitro tests which have correlation with bioavailability data in humans and can reasonably predict bioavailability data in humans; (c) in vivo tests in humans or other mammals, in which the appropriate acute pharmacological effect of an antibody (or its target) is determined as a function of time; and (d) determining the safety, efficacy, or bioavailability or bioequivalence of the antibody in a well-controlled clinical trial.

The biologically improved variants of the antibodies described herein can be based on existing reference antibodies having specificity for a target antigen (e.g., PD-1 or PD-L1) that have been altered such that, for example, the variants have a higher affinity for the target antigen and/or bind a different epitope, or have a more desirable therapeutic efficacy, expression, and/or biophysical characteristic, as compared to the reference antibody.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a small molecule PD-1/PD-L1 inhibitor having the formula:

in some embodiments, the PD-1 and/or PD-L1 inhibitor is a small molecule PD-1/PD-L1 inhibitor having formula (II)

Or a pharmaceutically acceptable salt thereof; wherein:

R¹selected from the group consisting of: halogen, C_5-8Cycloalkyl radical, C_6-10Aryl and thienyl, wherein, C_6-10Aryl and thienyl are optionally substituted with 1 to 5R^xSubstituent group substitution;

each R^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅and-S (O)₂NR^aR^bWherein each X¹Is C_1-4An alkylene group; each R^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^aAnd R^bMay be combined with a nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, wherein the five-or six-membered ring is optionally substituted by oxo; each R^cIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl and C_1-8A haloalkyl group; and optionally, when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five-, six-or seven-membered carbocyclic or heterocyclic ring, optionally substituted with 1 to 3 substituents independently selected from: halogen, oxo, C_1-8Haloalkyl and C_1-8Alkyl substituent substitution;

each R^2a、R^2bAnd R^2cIndependently selected from the group consisting of: H. halogen, -CN, -R^d、-CO₂R^e、-CONR^eR^f、-C(O)R^e、-OC(O)NR^eR^f、-NR^fC(O)R^e、-NR^fC(O)₂R^d、-NR^e-C(O)NR^eR^f、-NR^eR^f、-OR^e、-O-X²-OR^e、-O-X²-NR^eR^f、-O-X²-CO₂R^e、-O-X²-CONR^eR^f、-X²-OR^e、-X²-NR^eR^f、-X²-CO₂R^e、-X²-CONR^eR^f、-SF₅、-S(O)₂NR^eR^f、C_6-10Aryl and C_5-10Heteroaryl of which each X²Is C_1-4An alkylene group; each R^eAnd R^fIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^eAnd R^fMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O and S as ring members, and optionally substituted with oxo; each R^dIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group;

R³selected from the group consisting of: -NR^gR^hAnd C_4-12Heterocyclic group, wherein C_4-12Heterocyclyl is optionally substituted by 1 to 6R^ySubstitution;

each R^yIndependently selected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^j、-CONR^jR^k、-CONHC_1-6alkyl-OH, -C (O) R^j、-OC(O)NR^jR^k、-NR^jC(O)R^k、-NR^jC(O)₂R^k、CONOH、PO₃H₂、-NR^j-C_1-6alkyl-C (O)₂R^k、-NR^jC(O)NR^jR^k、-NR^jR^k、-OR^j、-S(O)₂NR^jR^k、-O-C_1-6alkyl-OR^j、-O-C_1-6alkyl-NR^jR^k、-O-C_1-6alkyl-CO₂R^j、-O-C_1-6alkyl-CONR^jR^k、-C_1-6alkyl-OR^j、-C_1-6alkyl-NR^jR^k、-C_1-6alkyl-CO₂R^j、-C_1-6alkyl-CONR^jR^kAnd SF₅，

Wherein R is^yC of (A)_1-6The alkyl moiety being optionally further substituted by OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted, wherein each R is^jAnd R^kIndependently selected from: hydrogen, optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8Alkyl, and optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8A haloalkyl group; or R when attached to the same nitrogen atom^jAnd R^kMay be combined with a nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and which is optionally substituted by oxo; each RⁱIndependently selected from the group consisting of: -OH, C_1-8Alkyl radical, C_2-8Alkenyl and C_1-8Haloalkyl, each of which may optionally be substituted with OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted;

R^gselected from the group consisting of: H. c_1-8Haloalkyl and C_1-8An alkyl group;

R^hselected from optionally CO₂H substituted-C_1-8Alkyl radical, C_1-8Haloalkyl, C_1-8alkyl-COOH, C_1-8alkyl-OH, C_1-8alkyl-CONH₂、C_1-8alkyl-SO₂NH₂、C_1-8alkyl-PO₃H₂、C_1-8alkyl-CONOH, C_1-8alkyl-NR^h1R^h2、-C(O)-C_1-8Alkyl, -C (O) -C_1-8alkyl-OH, -C (O) -C_1-8alkyl-COOH, C_3-10Cycloalkyl, -C_3-10cycloalkyl-COOH, -C_3-10cycloalkyl-OH, C_4-8Heterocyclyl radical, -C_4-8heterocyclyl-COOH, -C_4-8heterocyclyl-OH, -C_1-8alkyl-C_4-8Heterocyclyl radical, -C_1-8alkyl-C_3-10Cycloalkyl radical, C_5-10Heteroaryl, -C_1-8alkyl-C_5-10Heteroaryl group, C₁₀Carbocyclyl, -C_1-8alkyl-C_6-10Aryl radical, -C_1-8Alkyl- (C ═ O) -C_6-10Aryl radical, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkenyl, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkyl, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkynyl, -C_1-8Alkyl- (C ═ O) -NH-C_1-8Alkyl groups-COOH and-C_1-8Alkyl- (C ═ O) -NH-C_1-8alkyl-OH; or

R^hAnd N linked thereto is a mono-, di-or tripeptide comprising 1-3 natural amino acids and 0-2 unnatural amino acids, wherein,

the substituent for the alpha carbon of the unnatural amino acid is selected from the group consisting of: c_2-4Hydroxyalkyl radical, C_1-3Alkyl-guanidino and C_1-4An alkyl-heteroaryl group, which is a cyclic alkyl group,

the alpha carbon atom of each natural or unnatural amino acid is optionally further substituted with methyl, and

the terminal portion of the mono-, di-, or tripeptide is selected from the group consisting of: c (O) OH, C (O) O-C_1-6Alkyl and PO₃H₂Wherein, in the step (A),

R^h1and R^h2Each independently selected from the group consisting of: H. c_1-6Alkyl and C_1-4A hydroxyalkyl group;

R^hc of (A)_1-8The alkyl moiety is optionally further substituted with 1 to 3 substituents independently selected from the group consisting of: OH, COOH, SO₂NH₂、CONH₂、CONOH、COO-C_1-8Alkyl, PO₃H₂And optionally substituted by 1 to 2C_1-3Alkyl substituent substituted C_5-6(ii) a heteroaryl group, wherein,

R^hc of (A)₁₀Carbocyclyl, C_5-10Heteroaryl and C_6-10The aryl moiety is optionally substituted with 1 to 3 substituents independently selected from the group consisting of: OH, B (OH)₂、COOH、SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl radical, C_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H₂、C_1-4alkyl-COOH and phenyl and

R^hc of (A)_4-8Heterocyclyl and C_3-10Cycloalkyl moieties optionally substituted with 1 to 4R^wSubstituent group substitution;

each R^wThe substituents are independently selected from: c_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-COOH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H、OH、COO-C_1-8Alkyl, COOH, SO₂NH₂、CONH₂、CONOH、PO₃H₂And an oxo group;

R⁴selected from the group consisting of: O-C_1-8Alkyl, O-C_1-8Haloalkyl, O-C_1-8alkyl-R^z、C_6-10Aryl radical, C_5-10Heteroaryl, -O-C_1-4alkyl-C_6-10Aryl and-O-C_1-4alkyl-C_5-10Heteroaryl of which C is_6-10Aryl and C_5-10Heteroaryl is optionally substituted with 1 to 5R^zSubstitution;

each R^zIndependently selected from the group consisting of: halogen, CN, -R^m、-CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅、-S(O)₂RⁿR^p、-S(O)₂NRⁿR^pAnd a three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring, wherein the three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring is optionally substituted with 1 to 5R^tIn which each R is^tIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Halogenated alkyl, CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅and-S (O)₂NRⁿR^p；

Wherein each X³Is C_1-4An alkylene group; each RⁿAnd R^pIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atomⁿAnd R^pMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R^mIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group; and optionally, when two R are^zWhen the substituents are on adjacent atoms, they combine to form a fused five or six membered carbocyclic or heterocyclic ring, which carbocyclic or heterocyclic ring is optionally substituted by oxo;

n is 0, 1,2 or 3;

each R⁵Independently selected from the group consisting of: halogen, CN, -R^q、-CO₂R^r、-CONR^rR^s、-C(O)R^r、-OC(O)NR^rR^s、-NR^rC(O)R^s、-NR^rC(O)₂R^q、-NR^r-C(O)NR^rR^s、-NR^rR^s、-OR^r、-O-X⁴-OR^r、-O-X⁴-NR^rR^s、-O-X⁴-CO₂R^r、-O-X⁴-CONR^rR^s、-X⁴-OR^r、-X⁴-NR^rR^s、-X⁴-CO₂R^r、-X⁴-CONR^rR^s、-SF₅、-S(O)₂NR^rR^sWherein each X⁴Is C_1-4An alkylene group; each R^rAnd R^sIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^rAnd R^sMay combine with the nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group;

R^6aselected from the group consisting of: H. c_1-4Alkyl and C_1-4A haloalkyl group;

each R^6bIndependently selected from the group consisting of: F. c_1-4Alkyl, OR^u、C_1-4Haloalkyl, NR^uR^vWherein each R is^uAnd R^vIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^uAnd R^vMay combine with the nitrogen atom to form a five or six membered ring having 0 to 2 additional heteroatoms selected from N, O or S as ring members, and which is optionally substituted by oxo; and

m is 0, 1,2, 3 or 4.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor is selected from a compound or pharmaceutical composition disclosed in WO 2018/005374 filed by ChemoCentryx on 26/6/2017. The contents of which are incorporated herein for all purposes.

The PD-1 and/or PD-L1 inhibitors disclosed herein can be formulated to delay degradation of the compound or antibody or to minimize immunogenicity of the antibody. Various techniques are known in the art to address this goal.

In the combination therapies described herein, the CCR2antagonist can be formulated with other therapeutic agents or separately. The CCR2antagonist and other therapies will be formulated in suitable dosage unit formulations (alone or together), containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each mode of administration. It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, genetic characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

The biological product (e.g., an antibody) can comprise a pharmaceutical composition comprising one or more antibodies or fragments thereof and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers and/or adjuvants (such as preservatives, wetting agents, emulsifying agents and dispersing agents).

In some embodiments, if the therapeutic compound and agent are each provided in an amount that is subtherapeutic if provided alone or without the other. One skilled in the art will appreciate that "combination" may include combinations in therapy (i.e., two or more drugs may be administered as a mixture, or introduced into a subject at least at the same time or at least at different times but such that both are within the subject at the same time).

Likewise, the compounds, agents and compositions of the present invention may be used in combination with other drugs used to treat, prevent, inhibit or ameliorate cancer. Such other agents may be administered, by a route and in an amount commonly used therefor, either simultaneously or sequentially with a compound, agent or composition of the invention. When a compound, agent or composition of the invention is used contemporaneously with one or more other drugs, a pharmaceutical composition containing a drug other than a compound, agent or composition of the invention is preferred. Accordingly, the pharmaceutical compositions comprise, in addition to a compound, agent or composition of the invention, one or more other active ingredients or therapeutic agents.

Combination therapy includes co-administration of a CCR2antagonist and an additional therapeutic agent, sequential administration of a CCR2antagonist and an additional therapeutic agent, administration of a composition comprising a CCR2antagonist and an additional therapeutic agent 1 inhibitor, or simultaneous administration of separate compositions, wherein one composition comprises a CCR2antagonist and the other composition comprises an additional therapeutic agent.

Example IV

The following examples are intended to illustrate, but not limit, the claimed invention.

Materials & methods

Animals and cell lines. Female C57BL/6 mice (6-8 weeks old) were purchased from Charles River (Hollister, CA) and housed in an animal facility at the university of california davis division (UCDAVIS) at sakrauton, california. All animal experiments were performed according to the guidelines and approvals of the institutional animal care and use committee of UCDAVIS. The MBL2 cell line is an established cell line derived from Moloney MuLV-induced T cell lymphoma in C57BL/6 mice in which the gag gene was deleted from the genome to reduce virus-dependent immunogenicity. MBL2 cells were cultured in DMEM (Invitrogen, carlsbad, CA) containing 10% heat-inactivated FBS.

MBL2 tumors were established in mice. Methods for establishing mouse ear skin tumors have been previously described [28]. Briefly, PBS-washed MBL2 cells (4X 10 in 20. mu.l PBS) were injected into a 28g needle⁵) Injected into the dermis layer below the central dorsal surface of the ear and above the cartilage plane. Mice were then treated topically once with DNFB (1-fluoro-2, 4-dinitrobenzene, 0.5% in vehicle consisting of acetone and olive oil 4: 1(v/v), 10 μ l/ear) (Sigma), holy lous, CA) on the skin of the back ears. Maximum ear thickness was obtained using digital calipers, or tumor growth was assessed by removing the weight of the entire tumor-bearing ear from the ear baseline. The endpoint was determined based on the maximum allowable ear tumor size or local erosion and bleeding, which typically occurred within two weeks after implantation.

CCR2 antagonists and/or anti-PD 1 tumor therapy. High (6mg/ml) or low (2mg/ml) concentrations of the small molecule compound (compound 1), as well as vehicle controls, were provided by chemicentryx (Mountain View, Ca), a laboratory ready-to-use formulation. In therapeutic applications, oral administration via gavage of compound 1 or vehicle was initiated on the same day as MBL2 cell inoculation, typically two hours apart. After tumor implantation, Compound 1 was administered once daily (high dose 60mg/kg or low dose 20mg/kg) for two weeks. Mice were euthanized on day 3 or day 7 to analyze early immune responses to the treatment. For tumor treatment with anti-PD 1, MAb anti-mouse PD-1(CD279) and rat-IgG 2a (BioXcell, West Lebanon, NH) were injected three times a week into the body by IP (10 mg/kg per mouse). For combination therapy, the above single-dose regimen remains unchanged.

H & E and tissue immunochemistry. After surgical removal of mouse ear tumors at the base of the mouse ear, ear samples were cut into two parts along the long axis and placed in RNAlater for RNA extraction, or in 10% formalin for hematoxylin and eosin (H & E) staining or immunohistochemical staining with purified mouse antibodies (anti-CD 8 and anti-F4/80 from Protoyotech, san Diego, Calif.).

And (5) real-time quantitative PCR. RNA (< 2 μ g per sample) was converted to cDNA using a high capacity first strand cDNA kit (Qiagen). Real-time PCR was performed on a StepOne Plus real-time PCR system (Applied biosystems, carlsbad, california). QPCR primer pairs were purchased from Integrated DNA Technologies (Kolavian, Iowa).

CD8T cell depletion in tumor models. The day before tumor implantation, InVivo Plus anti-mouse CD8 α (clone 53-6.7) purchased from BioXcell was injected into mice by intraperitoneal route (250 μ g per injection). A second injection was given 7 days later at the same dose. To analyze the effect of CD8 depletion, ear tumor-inoculated mice were euthanized three days after the first dose. Cervical draining lymph nodes were collected and cell suspensions were isolated for flow cytometry analysis, including staining with FITC-anti-CD 8 (different clone 5H10-1, chinampa, san diego, california).

Flow cytometry for mouse ear tissue, lymph nodes and spleen. Anti-mouse CD45 (clone 30-F11), CD11b (M1/70), F4/80(BM8), Ly6G (1A8), Ly6C (HK1.4), IFN-. gamma. (XMG1.2) and CD8(5H10-1) antibodies were purchased from Lotudina (san Diego, Calif.). The ear or tumor tissue is digested to obtain a skin cell suspension as described [30 ]. Lymph nodes or spleens were directly minced and filtered through a cell strainer with 100 μm micron pores (Thermo Fisher Scientific), waltham, massachusetts). Red blood cells were removed from spleen samples using RBC lysis buffer (chinchow technique). Cells were incubated with brefeldin a and PMA/ionomycin for 4 hours and then stained intracellularly as described above. Flow cytometry was performed using Acuri C6 or LSR II (BD biosciences, San Jose, CA) in combination with FlowJo analysis software (Tree Star, San Carlos, CA).

And (5) carrying out statistical analysis. All data are expressed as mean ± SEM. Data were analyzed using GraphPad Prism version 6(GraphPad software, san diego, california). The mean and SEM of the data were simply compared using a two-sided student's t-test. A p-value <0.05 is considered statistically significant.

Example 1: in a murine model of Cutaneous T Cell Lymphoma (CTCL), small molecule CCR2 antagonists deplete tumor macrophages and stimulate CD8T cell accumulation (overview)

Tumor-associated macrophages (TAMs) recruited from blood monocytes play a key role in establishing an immunosuppressive Tumor Microenvironment (TME) that supports tumor growth. We have reported that high-grade cutaneous T cell lymphoma was established in the skin of syngeneic mice by injecting MBL 2T lymphoma cells in the skin of the ear followed by application of DNFB. In this model, macrophages play a key role in maintaining tumor growth. Therefore, we hypothesize that preventing monocytes (by inhibiting specific chemokine receptors) from entering the skin affects tumor development. Here, we investigated the effect of orally administered small molecule drugs (compound 1) that block CCR 2-mediated monocyte chemotaxis in this tumor model. After two consecutive days of compound 1 administration after tumor development, we measured (by flow cytometry) a significant depletion of macrophages in the skin (17.7% and 2.78% of total leukocytes, respectively, in vehicle and compound 1 treated mice). One week after treatment, neutrophilic abscesses and epidermal ulceration developed at the tumor sites in compound 1-treated mice, but not in vehicle-treated mice. Significant neutrophil increase was detected by flow cytometry in TME after compound 1 treatment. At two weeks, most mice in the control group were euthanized due to large tumors. However, due to the strong inflammatory response developed by the significantly larger number of CD8+ T cells within the tumor (identified by immunohistochemistry), the tumors treated with compound 1 became smaller and sometimes almost eradicated. Taken together, our data show a significant reduction in tumor macrophage accumulation in compound 1-treated mice, along with a reduction in tumor size in many animals and an increase in CD8+ T cells in TME. We propose therapeutic strategies based on CTCL that inhibit the CCR2 receptor and modulate the tumor microenvironment worthy of further exploration.

Example 2: compound 1(CCR2 antagonist) inhibits tumor progression in a cutaneous T cell lymphoma mouse model

We have previously reported a model of inflammation-dependent mouse T-cell lymphoma formed by implanting MBL2 cells into the subcutaneous skin followed by a single topical application of 2, 4-dinitro-1-fluorobenzene (DNFB) in the ear. Although in syngeneic mice, implantation of MBL2 cells alone into the subcutaneous ear did not result in tumor formation, presumably because DNFB-induced inflammation is often necessary for effective tumor formation. However, when a single dose of DNFB (a well studied contact allergen) was administered to the dorsal skin of mice immediately after tumor cell implantation, the resulting Tumor Microenvironment (TME) resulted in the generation of a reproducible tumor within two weeks. The use of DNFB induces a large number of inflammatory cells penetrating into TME, which mainly comprise myeloid cell populations, i.e. macrophages and neutrophils. By inducing macrophage "suicide" using clodronate liposomes, we have shown that macrophages in the MBL2/DNFB model contribute to tumor growth [29 ]. Therefore, we hypothesized that compounds targeting chemokine receptor CCR2 for blocking monocyte recruitment and macrophage differentiation in TME might also reduce the growth of cutaneous T cell lymphoma tumors.

Compound 1 is an orally bioavailable CCR2 antagonist. Plasma concentrations of compound 1 in mice were closely related to the fed dose after administration at two different doses (20mg/kg or 60mg/kg) by daily oral gavage (fig. 2A). In addition, neither of these dosing regimens resulted in significant (> 20%) changes in mouse body weight (fig. 2B), indicating that the drug was well tolerated. For the experimental treatment protocol, we administered compound 1 daily by oral gavage starting on the same day of tumor implantation (fig. 1 a). Treatment of tumors of the ear in mice with two different doses of compound 1 showed a significant reduction in tumor growth, but not in mice treated with vehicle (fig. 1C). Both ear thickness and ear weight (measured immediately after euthanasia) were significantly reduced with compound 1 compared to untreated or vehicle-treated controls (fig. 1C). Thus, compound 1 blocks tumor growth in an inflammation and macrophage dependent model of T cell lymphoma.

Example 3: CCR2 after Compound 1treatment⁺Macrophages are specific in the Tumor Microenvironment (TME)Is consumed by nature

As we have previously demonstrated, in the MBL2/DNFB model, DNFB induces inflammatory TME in the ear skin, i.e., within a short two days, redness, edema and rapid accumulation of large numbers of inflammatory cells in the ear [28]]. To reveal the mechanism by which compound 1 reduces tumor growth in mice, we examined mice treated with compound 1 two days after tumor implantation. Flow cytometry analysis of cell suspensions from ear tumors revealed that CD11b was treated with compound 1⁺/F4/80⁺The level of macrophages was significantly reduced, including the percentage and absolute number of viable cells calculated by whole ear (fig. 3A)&B) In that respect Since both flow data and tumor measurements indicate that higher doses of compound 1 produced better therapeutic effect without side effects, we used 60mg/kg per day as the standard dose in the subsequent experiments.

Other bone marrow-derived subpopulations known to be closely related in immune function to macrophages accumulate in TME. We wanted to know whether Compound 1 specifically targets the CD11b and F4/80 positive macrophages detected above. By flow cytometry binding surface markers to single cell suspensions from whole ear tissue (i.e., CD11b, F4/80, Ly6G, Ly6C and CCR2), we clearly seen the presence of two types of cells controlling the myeloid cell population gated on CD11b (fig. 3C). However, F4/80 positive cells were the cell type targeted by Compound 1. This population co-expressed Ly6C and CCR2 (a functional target for compound 1). Another major cell population consisted of CD11b and Ly6G positive cells, which also co-expressed Ly6C, but not CCR2 (fig. 3C). Although these cells display neutrophil markers, we call them neutrophil-like cells because their potential immature nature and characteristics in establishing a tumor microenvironment are similar to MDSCs (myeloid-derived suppressor cells). As shown in fig. 3C, neutrophil accumulation was not blocked by compound 1; in contrast, their relative content is increased due to depletion of macrophages by CCR2 antagonism.

Example 4: compound 1treatment can enhance intratumoral inflammation

During the course of tumor formation, ear skin inflammation was significantly enhanced in mice treated with compound 1, with the skin being redder and scalier than in control mice. Histological examination of the tissues starting from day 7 revealed that the surface of the ear on the back side (i.e. the side exposed to DNFB) showed surface ulceration, scaling and accumulation of significant inflammatory infiltrates under the microscope (fig. 4A). IHC staining with anti-F4/80 confirmed the substantial absence of macrophages in the TME (FIG. 4B). Flow cytometry analysis of tissues from the same time point showed a significant increase in neutrophil-like cells, consistent with histological performance (fig. 4C). Notably, not only the percentage but also the total number increased, indicating that recruitment of neutrophil-like cells to TME was accompanied by macrophage depletion. Thus, treatment with compound 1 resulted in tumor cell necrosis and increased neutrophil-like cells in TME with a small number of F4/80+ macrophages.

Example 5: compound 1treatment can alter cytokines and immune biomarkers in TME

To further understand the potential mechanism of compound 1-mediated tumor inhibition, we quantified cytokines and chemokines known to be involved in anti-tumor immunity in compound 1-treated tumors. Interestingly, by treatment with compound 1, IFN- γ -induced chemokines, CXCL10 and CXCL11 in the ear were all significantly increased at the mRNA level. IL-12 (another Th1 marker) was found to be increased in the ears of compound 1 treated mice. We seen a sustained upregulation of granzyme B, another indication of activation of the anti-tumour cytotoxic pathway (figure 5A). In contrast, expression of the representative Th2 cytokines IL-10 and TGF-. beta.were similarly expressed between compound 1-treated mice and the control group (FIG. 6).

Additional analysis of gene expression indicated that several major inflammatory cytokines (e.g., IL-17a, IL-1 β, and IL-6) were upregulated to varying degrees in Compound 1-treated mice. During CCR2 antagonism in TME, upregulation of CCL2 (ligand for CCR2) and its closely related chemokine CCL7 likely reflected increased transcription of CCL2 in the context of effective CCR2 inhibition (fig. 5B). The last group of significantly increased biomarkers in TME after compound 1treatment was confirmed to be neutrophil chemotaxis and biomarkers, i.e., CXCL1/2 and S100a8/9 (fig. 5C), consistent with the recruitment of neutrophil-like cells shown by flow cytometry (fig. 4C).

Example 6: macrophage blockade of CD8T cells in TME mediates antitumor activity

Next, we investigated whether compound 1 requires CD8T cells to effectively block tumor growth. Tumor tissue was collected from tumor-bearing mice that received two weeks of treatment. Few CD8T cells were observed in untreated and vehicle-treated tumors by IHC staining (fig. 7A). However, compound 1treatment significantly increased CD8 in TME in a dose-dependent manner⁺Number of T cell infiltrates (fig. 7A).

Next, we administered neutralizing CD8 antibody by IP injection simultaneously with compound 1treatment (fig. 7B). Three days after the first injection of the depleting anti-CD 8 antibody, we found that there were few CD3+/CD8+ T cells in the cervical draining lymph nodes of antibody-treated mice (fig. 7C). Two weeks after anti-CD 8 treatment, measurements of the terminal ear tumor size again indicated that compound 1treatment inhibited tumor growth as shown (green triangle), while anti-CD 8 abolished this effect (similar to the levels in the vehicle-treated group) (fig. 7D). In the DNFB-MBL2 model, the size of ipsilateral cervical draining lymph nodes was closely related to lymph node metastasis. As shown in fig. 7E, the reduction in size of cervical LN treated with compound 1 was reversed by CD8T cell depletion. Thus, treatment with compound 1 requires the presence of CD8+ T cells to effectively reduce tumor growth and LN metastasis.

Example 7: macrophage blockade in conjunction with anti-PD 1 to limit MBL2 tumor growth

The role of PD1 in the immune escape of cancer is well established in terms of tumor cells or antigen presenting cells (e.g., macrophages) expressing PD-L1 and interacting with PD-1 positive CD8-T cells to render them unresponsive to anti-tumor activity. Thus, inhibitors that block the interaction between PD-1 and PD-L1 may enhance T cell responses, referred to as immune checkpoint blockade. MBL2 tumors formed in mice showed a significant increase in PD-L1 compared to MBL2 cells cultured in vitro (fig. 7A). Similar to the results for compound 1, antibody blockade of the PD-1/PD-L1 axis in this MBL2 model was effective in delaying tumor growth, but not in eliminating tumors (fig. 9A-B). This prompted us to combine single drugs to achieve better anti-tumor effects.

For the combination therapy, mice were treated with 10mg/kg of anti-PD 1 every other day, starting on the same day as the first compound 1treatment and tumor implantation (fig. 8B). Mice were euthanized after two weeks of treatment and we analyzed spleens from each group and found that the number of IFN- γ producing CD8-T cells was significantly increased in the group treated with the combination of compound 1 and anti-PD 1, indicating that these mice exhibited stronger anti-tumor immunity (fig. 8C). In examining ear tumor size, we found that combination treatment significantly inhibited the growth of ear tumors compared to isotype and vehicle treated controls, but not anti-PD 1 and vehicle treatment. According to our past long-term observation of the MBL2/DNFB model, i.e. more than 6 weeks after tumor implantation, mice with ear thickness less than 1mm rarely, if at all, develop tumors within two weeks. As shown by the horizontal dashed lines in the figure, the tumor thickness was less than 1mm in nearly 75% of mice from the combination treatment group, indicating long-term tumor clearance (fig. 8D).

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application controls.

Claims

1. A method of treating a solid tumor comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

2. The method of claim 1, wherein the CCR2antagonist has a structure according to the formula:

A is C (R)⁵)(R⁶) Or N (R)⁵)

R¹selected from the group consisting of: aryl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl, wherein the heteroaryl moiety has 1-3 heteroatoms selected from N, O and S as ring members; and wherein said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 5R^xSubstituent group substitution;

R²selected from the group consisting of: H. c_1-8Alkyl radical, C_3-8Cycloalkyl radical, C_3-8cycloalkyl-C_1-4Alkyl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl, wherein the heteroaryl moiety has 1-3 heteroatoms selected from N, O and S as ring members; and wherein said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 4R^xSubstituent group substitution;

R³selected from the group consisting of: H. c_1-8Alkyl radical, C_3-8CycloalkanesRadical and C_3-8cycloalkyl-C_1-4Alkyl, each of which is optionally substituted with 1 to 3R^ySubstituent group substitution;

or optionally, R⁵And R⁶Combined to form a spirocyclic 5-or 6-membered cycloalkyl ring, optionally unsaturated, and optionally substituted with 1 to 4R^zA fused aryl group substituted with a substituent;

each R^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aC(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-NR^aR^b、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅、-S(O)₂NR^aR^bAnd 5-or 6-membered aryl or heteroaryl, wherein each X¹Is C_1-4An alkylene group; each R^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^aAnd R^bMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by oxo; each R^cIndependently selected from: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; and optionally, when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five or six membered carbocyclic ring, and wherein the aryl or heteroaryl group is optionally substituted with 1 to 3 members selected from the group consisting of: halogen, hydroxy, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Haloalkyl and C_1-4A haloalkoxy group;

each R^zIndependently selected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-OC(O)NR^gR^h、-NR^hC(O)R^g、-NR^hC(O)₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-S(O)₂NR^gR^h-、-X¹-R^j、-X¹-NR^gR^h、-X¹-CONR^gR^h、-X¹-NR^hC(O)R^g、-NHR^j、-NHCH₂R^jAnd tetrazole; wherein each R is^gAnd R^hIndependently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^gAnd R^hMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by one or two oxo groups; each RⁱIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; each R^jSelected from the group consisting of: c_3-6Cycloalkyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl and tetrahydropyranyl.

3. The method of claim 1, wherein said CCR2antagonist is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the CCR2antagonist has the structure shown in compound 1:

or a pharmaceutically acceptable salt thereof.

5. The method of claim 1, wherein the CCR2antagonist has the structure shown in compound 2:

or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the CCR2antagonist has the structure shown in compound 3:

or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1-6, wherein the solid tumor is selected from the group consisting of: skin cancer, brain cancer, breast cancer, triple negative breast cancer, bladder cancer, bone cancer, colorectal cancer, lung cancer, kidney cancer, liver cancer, stomach cancer, prostate cancer, sarcoma, melanoma, carcinoma, and lymphoma.

8. The method of any one of claims 1-6, wherein the solid tumor is selected from the group consisting of: skin cancer, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, and lymphoma.

9. The method of any one of claims 1-6, wherein the solid tumor is a lymphoma.

10. The method of claim 9, wherein the lymphoma is cutaneous T-cell lymphoma (CTCL).

11. The method of any one of claims 1 to 10, wherein the treatment results in a reduction in tumor size compared to an individual not administered the CCR2 antagonist.

12. The method of any one of claims 1 to 10, wherein the treatment blocks tumor growth.

13. The method of any one of claims 1 to 10, wherein said treatment eradicates said solid tumor.

14. The method of any one of claims 1 to 13, wherein the CCR2antagonist is administered orally.

15. The method of any one of claims 1 to 14, further comprising administering one or more additional therapeutic agents.

16. The method of claim 15, wherein the one or more additional therapeutic agents is an immune checkpoint inhibitor.

17. The method of claim 16, wherein the immune checkpoint inhibitor is a PD-1 and/or PD-L1 inhibitor.

18. The method of claim 17, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, biosimilar thereof, biological modification thereof, and biological equivalent thereof.

19. The method of claim 17, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: nivolumab, pembrolizumab, and pidilizumab.

20. The method of claim 17, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: duvivumab, alemtuzumab, Ablumumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, CA-327, STI-1014, KY-1003, biological analogs thereof, biological modifiers thereof and biological equivalents thereof.

21. The method of claim 17, wherein the PD-1 and/or PD-L1 inhibitor is a compound of formula (II)

Or a pharmaceutically acceptable salt thereof; wherein:

each R^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅and-S (O)₂NR^aR^bWherein each X¹Is C_1-4An alkylene group; each R^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^aAnd R^bCan be combined with a nitrogen atom to form a compound having 0 to 2 additional atoms selected from N, O or SA five-or six-membered ring with a heteroatom as a ring member, wherein the five-or six-membered ring is optionally substituted by oxo; each R^cIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl and C_1-8A haloalkyl group; and optionally, when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five-, six-or seven-membered carbocyclic or heterocyclic ring, optionally substituted with 1 to 3 substituents independently selected from: halogen, oxo, C_1-8Haloalkyl and C_1-8Alkyl substituent substitution;

Wherein R is^yC of (A)_1-6The alkyl moiety being optionally further substituted by OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted, wherein each R is^jAnd R^kIndependently selected from: hydrogen, optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8Alkyl, and optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8A haloalkyl group; or R when attached to the same nitrogen atom^jAnd R^kMay be combined with a nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, andoptionally substituted with oxo; each RⁱIndependently selected from the group consisting of: -OH, C_1-8Alkyl radical, C_2-8Alkenyl and C_1-8Haloalkyl, each of which may optionally be substituted with OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted;

a substituent of the unnatural amino acid having an alpha carbon selected from the group consisting of: c_2-4Hydroxyalkyl radical, C_1-3Alkyl-guanidino and C_1-4An alkyl-heteroaryl group, which is a cyclic alkyl group,

each R^zIndependently selected from the group consisting of: halogen, -CN, -R^m、-CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅、-S(O)₂RⁿR^p、-S(O)₂NRⁿR^pAnd a three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring, wherein the three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring is optionally substituted with 1 to 5R^tIn which each R is^tIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl, -CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅and-S (O)₂NRⁿR^p；

n is 0, 1,2 or 3;

each R⁵Independently selected from the group consisting of: halogen, -CN, -R^q、-CO₂R^r、-CONR^rR^s、-C(O)R^r、-OC(O)NR^rR^s、-NR^rC(O)R^s、-NR^rC(O)₂R^q、-NR^r-C(O)NR^rR^s、-NR^rR^s、-OR^r、-O-X⁴-OR^r、-O-X⁴-NR^rR^s、-O-X⁴-CO₂R^r、-O-X⁴-CONR^rR^s、-X⁴-OR^r、-X⁴-NR^rR^s、-X⁴-CO₂R^r、-X⁴-CONR^rR^s、-SF₅、-S(O)₂NR^rR^sWherein each X⁴Is C_1-4An alkylene group; each R^rAnd R^sIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^rAnd R^sMay combine with the nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and which is optionally substituted by oxo; each R^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group;

each R^6bIndependently selected from the group consisting of: F. c_1-4Alkyl, OR^u、C_1-4Haloalkyl, NR^uR^vWherein each R is^uAnd R^vIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^uAnd R^vMay combine with the nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and which is optionally substituted by oxo; and

m is 0, 1,2, 3 or 4.

22. A method of increasing the number of CD8+ T cells in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

23. The method of claim 22, wherein said CCR2antagonist has the structure shown below:

or a pharmaceutically acceptable salt, hydrate, stereoisomer or rotamer thereof; wherein

A is C (R)⁵)(R⁶) Or N (R)⁵)

R¹selected from the group consisting of: aryl, aryl-C_1-4Alkyl, heteroaryl and heteroaryl-C_1-4Alkyl radicalWherein the heteroaryl moiety has 1-3 heteroatoms selected from N, O and S as ring members; and wherein said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 5R^xSubstituent group substitution;

or optionally, R¹And R²Combine with the nitrogen atom to which each is attached to form a 6-to 11-membered monocyclic or fused bicyclic heterocyclic or heteroaryl ring, wherein-NR¹R²Optionally further substituted with 1 to 4R^xSubstituent group substitution;

or optionally，R⁵And R⁶Combined to form a spirocyclic 5-or 6-membered cycloalkyl ring, optionally unsaturated, and optionally substituted with 1 to 4R^zA fused aryl group substituted with a substituent;

each R^zIndependently selected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-OC(O)NR^gR^h、-NR^hC(O)R^g、-NR^hC(O)₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-S(O)₂NR^gR^h、-X¹-R^j、-X¹-NR^gR^h、-X¹-CONR^gR^h、-X¹-NR^hC(O)R^g、-NHR^j、-NHCH₂R^jAnd tetrazole; wherein each R is^gAnd R^hIndependently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^gAnd R^hMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by one or two oxo groups; each RⁱIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; each R^jSelected from the group consisting of: c_3-6CycloalkanesPhenyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuryl, and tetrahydropyranyl.

24. The method of claim 22, wherein said CCR2antagonist is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

25. The method of claim 22, wherein the CCR2antagonist has the structure shown in compound 1:

or a pharmaceutically acceptable salt thereof.

26. The method of claim 22, wherein the CCR2antagonist has the structure shown in compound 2:

or a pharmaceutically acceptable salt thereof.

27. The method of claim 22, wherein the CCR2antagonist has the structure shown in compound 3:

or a pharmaceutically acceptable salt thereof.

28. A method of reducing macrophage numbers in a solid tumor microenvironment, comprising administering an effective amount of a chemokine receptor 2(CCR2) antagonist.

29. The method of claim 28, wherein the CCR2antagonist has the structure shown below:

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a stereoisomer thereof, or a rotamer thereof; wherein

A is C (R)⁵)(R⁶) Or N (R)⁵)

or optionally, R¹And R²Combined with the nitrogen atom to which it is attached to form a 6-to 11-membered monocyclic or fused bicyclic heterocyclic or heteroaryl ring, wherein-NR¹R²Optionally further substituted by 1 to 4R^xSubstituent group substitution;

R³selected from the group consisting of: H. c_1-8Alkyl radical, C_3-8Cycloalkyl and C_3-8cycloalkyl-C_1-4Alkyl, each of which is optionally substituted with 1 to 3R^ySubstituent group substitution;

each R^zIndependently of each otherSelected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-OC(O)NR^gR^h、-NR^hC(O)R^g、-NR^hC(O)₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-S(O)₂NR^gR^h、-X¹-R^j、-X¹-NR^gR^h、-X¹-CONR^gR^h、-X¹-NR^hC(O)R^g、-NHR^j、-NHCH₂R^jAnd tetrazole; wherein each R is^gAnd R^hIndependently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^gAnd R^hMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, which may be optionally substituted by one or two oxo groups; each RⁱIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl and C_3-6A cycloalkyl group; each R^jSelected from the group consisting of: c_3-6Cycloalkyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl and tetrahydropyranyl.

30. The method of claim 28, wherein said CCR2antagonist is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

31. The method of claim 28, wherein the CCR2antagonist has the structure shown in compound 1:

or a pharmaceutically acceptable salt thereof.

32. The method of claim 28, wherein the CCR2antagonist has the structure shown in compound 2:

or a pharmaceutically acceptable salt thereof.

33. The method of claim 28, wherein the CCR2antagonist has the structure shown in compound 3:

or a pharmaceutically acceptable salt thereof.

34. A method of treating Cutaneous T Cell Lymphoma (CTCL), the method comprising administering to a subject in need thereof an effective amount of a chemokine receptor 2(CCR2) antagonist.

35. The method of claim 34, wherein the subject in need thereof is a stage IA CTCL.

36. The method of claim 34, wherein the subject in need thereof is a stage IB CTCL.

37. The method of claim 34, wherein the subject in need thereof is stage IIA CTCL.

38. The method of claim 34, wherein the subject in need thereof is stage IIB CTCL.

39. The method of claim 34, wherein the subject in need thereof is stage III CTCL.

40. The method of claim 34, wherein the subject in need thereof is stage IV CTCL.

41. The method of any one of claims 34 to 40, wherein the CCR2antagonist has the structure shown below:

A is C (R)⁵)(R⁶) Or N (R)⁵)

or optionally, R¹And R²Combined with the nitrogen atom to which they are attached to form a 6-to 11-membered monocyclic or fused bicyclic heterocycle or heteroarylBasic ring, wherein-NR¹R²Optionally further substituted with 1 to 4R^xSubstituent group substitution;

42. The method of any one of claims 34 to 40, wherein the CCR2antagonist is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

43. The method of any one of claims 34 to 40, wherein the CCR2antagonist has the structure shown in Compound 1:

or a pharmaceutically acceptable salt thereof.

44. The method of any one of claims 34 to 40, wherein the CCR2antagonist has the structure shown in Compound 2:

or a pharmaceutically acceptable salt thereof.

45. The method of any one of claims 34 to 40, wherein the CCR2antagonist has the structure shown in Compound 3:

or a pharmaceutically acceptable salt thereof.

46. The method of any one of claims 34 to 45, further comprising administering one or more additional therapeutic agents.

47. The method of claim 46, wherein the one or more additional therapeutic agents is an immune checkpoint inhibitor.

48. The method of claim 47, wherein the immune checkpoint inhibitor is a PD-1 and/or PD-L1 inhibitor.

49. The method of claim 48, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, biosimilar thereof, biological modification thereof, and biological equivalent thereof.

50. The method of claim 48, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: nivolumab, pembrolizumab, and pidilizumab.

51. The method of claim 48, wherein the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of: duvivumab, alemtuzumab, Ablumumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, CA-327, STI-1014, KY-1003, biological analogs thereof, biological modifiers thereof and biological equivalents thereof.

52. The method of claim 48, wherein the PD-1 and/or PD-L1 inhibitor is a compound of formula (II)

Or a pharmaceutically acceptable salt thereof; wherein:

each R^2a、R^2bAnd R^2cIndependently selected from the group consisting of: H. halogen, -CN, -R^d、-CO₂R^e、-CONR^eR^f、-C(O)R^e、-OC(O)NR^eR^f、-NR^fC(O)R^e、-NR^fC(O)₂R^d、-NR^e-C(O)NR^eR^f、-NR^eR^f、-OR^e、-O-X²-OR^e、-O-X²-NR^eR^f、-O-X²-CO₂R^e、-O-X²-CONR^eR^f、-X²-OR^e、-X²-NR^eR^f、-X²-CO₂R^e、-X²-CONR^eR^f、-SF₅、-S(O)₂NR^eR^f、C_6-10Aryl and C_5-10Heteroaryl of which each X²Is C_1-4An alkylene group; each R^eAnd R^fIndependent of each otherIs selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^eAnd R^fMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O and S as ring members, and optionally substituted with oxo; each R^dIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group;

Wherein R is^yC of (A)_1-6The alkyl moiety being optionally further substituted by OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted, wherein each R is^jAnd R^kIndependently selected from: hydrogen, optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8Alkyl, and optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by H_1-8A haloalkyl group; or R when attached to the same nitrogen atom^jAnd R^kMay be combined with a nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each RⁱIndependently selected from the group consisting of: -OH, C_1-8Alkyl radical, C_2-8Alkenyl and C_1-8Haloalkyl, each of which may optionally be substituted with OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted;

the unnatural amino acid has a substituent at the alpha carbon selected from the group consisting of: c_2-4Hydroxyalkyl radical, C_1-3Alkyl-guanidino and C_1-4An alkyl-heteroaryl group, which is a cyclic alkyl group,

n is 0, 1,2 or 3;

each R⁵Independently selected from the group consisting of: halogen, -CN, -R^q、-CO₂R^r、-CONR^rR^s、-C(O)R^r、-OC(O)NR^rR^s、-NR^rC(O)R^s、-NR^rC(O)₂R^q、-NR^r-C(O)NR^rR^s、-NR^rR^s、-OR^r、-O-X⁴-OR^r、-O-X⁴-NR^rR^s、-O-X⁴-CO₂R^r、-O-X⁴-CONR^rR^s、-X⁴-OR^r、-X⁴-NR^rR^s、-X⁴-CO₂R^r、-X⁴-CONR^rR^s、-SF₅、-S(O)₂NR^rR^sWherein each X⁴Is C_1-4An alkylene group; each R^rAnd R^sIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or R when attached to the same nitrogen atom^rAnd R^sMay combine with the nitrogen atom to form a five or six membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group;

m is 0, 1,2, 3 or 4.